Genome-wide analysis of mechanosensitive channel of small conductance ( MscS )-like gene family in common bean

Mechanosensitive (MS) ion channels are transmembrane proteins that open and close in response to mechanical forces produced by osmotic pressure, sound, touch and gravity. In plants, MS have an important role in different biological processes like gravity detection, maintenance of plastid shape and size, lateral root emergence, growth of pollen tube, and plant-pathogen interactions. In this study, homologous mechanosensitive channel of small conductance (MscS)-like gene family in common bean was identified. Nine Phaseolus vulgaris MscS-like (PvMSL) genes were found to be distributed on five chromosomes. A complete overview of PvMSL genes in common bean is presented, including gene structures, chromosome locations, phylogeny, protein motifs and expression pattern. Subcellular localization predictions of PvMSL family revealed their location to plasma and chloroplast membrane. Phylogenetic analysis of nine PvMSL proteins resulted in two main classes. The predicted gene structure, conserved motif, domain and presence of transmembrane regions in each PvMSL strongly supported their identity as members of MscS-like gene family. Four duplicate events of PvMSL genes were discovered in P. vulgaris chromosomes, and tandem and segmental duplication may cause the expansion of PvMSL genes. Furthermore, PvMSL genes displayed differential expression patterns in tissues and organs. This is the first step towards genome-wide analyses of MSL genes in common bean. Thus, the data obtained in this study provide resources to select candidate genes for future functional analyses that will help understand plant growth, development, and function of MSL gene family in P. vulgaris.

. Moreover, they respond to signals associated with plant development processes like emergence of lateral roots, growth of pollen tube, damages to the cell wall, and plant-pathogen interactions (Lucas et al., 2013;Appel and Cocroft, 2014;Jayaraman et al., 2014).In many of these cases, the application of a mechanical stimulus results in a quick explosion of the ion flow which can be attributed to mechanosensitive (MS) ions due to the response speed (Fasano et al., 2002;Jaffe at al., 2002;Monshausen and Haswell, 2013).Some responses are rapid, such as regulation of volume and turgor pressure of guard cells and closing of Venus's flytrap leaf on its prey (Cosgrove and Hedrich, 1991;Haswell and Meyerowitz, 2006;Furuichi et al., 2008b).Other responses to mechanical perturbation are growth-related and more slowly like repeated tactile stimulation of Arabidopsis seedlings which causes late flowering and short inflorescences (Braam and Davis, 1990) and Arabidopsis roots that alter their course through the soil to avoid a barrier (Massa and Gilroy, 2003).It has recently been shown that at least some mechanical stimuli trigger rapid molecular events that are then transduced into a necessarily slower growth response.In Arabidopsis, cytoplasmic alkalinization of root gravi-responsive cells occurs within 2 min of gravity stimulation and touch can elicit immediate Ca 2+ transients in root cells (Legue et al., 1997;Fasano et al., 2001).
The MS channels, formed by transmembrane proteins, work as mechano-electric transductors where mechanical stimuli are converted into electrical or chemical signals in live cells that perform numerous cellular processes associated with mechanosensory transduction (Kloda and Martinac, 2002).The first MSs were identified during the electrophysiological characterization of the plasmatic membrane of Escherichia coli (Martinac et al., 1987;Sukharev et al., 1993;Berrier et al., 1996).The MS channels were classified according to their ion conductance and the main ones are: Mechanosensitive Channels of Large Conductance (MscL) and Mechanosensitive Channels of Small Conductance (MscS) (Edwards et al., 2012;Cox et al., 2015).The MscL present conductance (larger than 2.4 nS), open undermembrane high tension and present rapid kinectic (Perozo, 2006).Each MscL subunit is composed of a polypeptide of 136 amino acids with two transmembrane helices, TM1 and TM2 (Sukharev et al., 1994(Sukharev et al., , 1999)).A 3.5 Å crystalline structure in TbMsLC ortholog (151amino acids) in Mycobacterium tuberculosis revealed a homo pentamer in an apparently closed state (Chang et al., 1998).On the other hand, MS of small conductance is a 0.8 to 1-nS channel opened by moderate pressure (Perozo, 2006).EcoMsSC structure of the channel from E. coli (286 amino acids) was originally resolved at 3.9 Å (Bass et al., 2002) and recently at 3.45 Å in an open configuration (Wang et al., 2008).In bacterial systems and animals, MS ion channels mediate the perception of pressure, touch and sound.
Although plants respond to a wide variety of Souza et al. 581 mechanical stimulli, the molecular nature of transmembrane protein mechanical perception in vegetal system has been little studied (Hedrich, 2012).MS ion channels were characterized in Arabidopsis thaliana and Oriza sativa model plants (Haswell and Meyerowitz, 2006;Saddhe and Kumar, 2015).In Arabidopsis and rice, ten and five MscS homologs, respectively, were identified and classified into two main classes, Class I and Class II.There has been no study done on Phaseolus vulgaris to identify MscS homologs.
Common bean (P.vulgaris) is a main socially important crop and is a major source of protein and essential nutrients.Common bean is the most consumed legume in the worldwide (Schmutz et al., 2014).Brazil is the largest producer with an average annual production of 3.5 million tons (MAPA, 2015).However, the grain yield in Brazil is considered low and several factors are related to this, as the adverse effects of climate conditions, as well as the occurrence of pests and diseases (Beebe et al., 2013).
Therefore, knowing the importance of MS ion channels of small conductance in plant development and the important role of MscS genes to overcome different stresses, and considering lack information of MscS in P. vulgaris, this study aimed to identify homologous MscSlike (MSL) genes in common bean and analyze their structure, subcellular location, phylogenetic relationship, expression pattern and chromosomal distribution.Our analysis may contribute to select candidate genes for future functional analyses that will aid researchers in understanding plant growth, development and function of MSL gene family in P. vulgaris.(Altschul et al., 1997).The sequences were compared with other sequences deposited in GenBank database, utilizing BlastP and BlastX programs (National Center for Biotechnology Information, NCBI; http://www.ncbi.nlm.nih.gov) to confirm their identity.The initially collected sequences, whose ORFs were incomplete, were excluded from the analyses.The protein sequences were aligned utilizing Clustal Omega algorithm version 2.0.3 (Sievers et al., 2011) and the redundant inputs were removed.

Characteristics of predicted proteins, transmembrane regions and subcellular location of MSL genes in P. vulgaris
The physical and chemical characteristics of MSL proteins in bean were calculated by ProtParam tool (http://web.expasy.org/protparam),including the number of amino

Phylogenetic analysis
Phylogenetic analysis was done by aligning the protein sequences utilizing Clustal Omega algorithm, version 2.0.3 (Sievers et al., 2011).The phylogenetic tree was built by Maximum-Likelihood (ML) method using pair-wise deletion with the help of MEGA program, version 6.06 (Tamura et al., 2013).1,000 bootstrap replicates were utilized to test analysis confidence.

Genome structure
Complete sequences of genomic DNA and coding sequences CDS, corresponding to each gene using Gene Structure Display Server 2.0 (GSDS) (http://gsds.cbi.pku.edu.cn/), were used to identify the position of introns and the organization of exon/intron in genes.
Annotation, duplication and synteny of MSL genes in P. vulgaris genome P. vulgaris MSL genes (PvMSL) were mapped in bean chromosome according to their genome coordinates.The genes were plotted in chromosomes using MapChart software and data on their physical location are avaialble in Phytozome.Duplications of PvMSL genes considered as parameters 50% identity and 80% coverage, adapted from Lopes-Caitar et al. (2013).Plant Genome Duplication Database (PGDD; http://chibba.agtec.uga.edu/duplication/) was used to search for orthologous genes in P. vulgaris and Arabidopsis.The synteny map was displayed using Circos software (http://circos.ca/)(Krzywinski et al., 2009).

Identification and classification of MSL genes in the common bean (P. vulgaris) genome
In order to identify MSL gene family in common bean genome, the amino acid sequence PF00925 and a Hidden Markov Model profile of MSL protein were used to perform a BLASTP search against the Common Bean databases v1.1 (Phytozome v10.3: http://www.phytozome.net).In this study, a total of nine genes encoding putative proteins from a MS ion channel of small conductance were identified from the whole genome and were named PvMSL1 to PvMSL9.Nine genes encoding MSL family were identified in P. vulgaris, while different numbers of genes were identified in other plant species (Table 1).The number of genes in MSL family identified in common bean was similar when compared with tomato (8), smaller than in Arabidopsis (10) and soybeans ( 14), and larger than in rice (6) and maize (5).Generally, the number of MSL genes distribution in dicotyledons is nearly double than in monocotyledons, possibly due to the higher rate of genome duplication events in the former.In Arabidopsis, Saddhe and Kumar (2015) attributed this to four different major genome duplication events over 100 to 200 million years ago.Moreover, the number of MSL genes in P. vulgaris is not associated with the size of the genome itself.Although P. vulgaris has a much larger genome (580 Mb) (Schmutz et al., 2014) than Arabidopsis (145 Mb) (Huala et al., 2001), the structure and phylogenic arrangements are quite similar.Indeed, the number of MSL genes in monocotyledons as well as in dicotyledonous seemed to be quite similar, despite the different sizes of the genomes (Table 1).In fact, any irregular distribution of genes in MSL family among different plant species may have evolved from genomic losses or duplication events of a common ancestor, contributing to the expansion of or restriction on the number of these genes.All of the putative MSL proteins were divided into two classes (that is, I and II).The members of Class I resembled prokaryotic MsSC channels, and the ones of Class II form independent plant/fungus lineages (Pivetti et al., 2003;Haswell, 2007).Table 1 summarizes MSL protein classification of common bean genome and other plant species.Classes I and II had four and five members, respectively.According to Pivetti et al. (2003)  resemble TM3 of MsSC.Similar motifs have been reported in bacterial MscS channel systems (Pivetti et al., 2003;Saddhe and Kumar, 2015).Proteins in Class II showed the presence of consensus sequence F(X3)P(X3)GD(X10-14)V(X20-21)PN(X7)IXNXXR at the C-terminal TM domain in P. vulgaris (PvMSL5, PvMSL6, PvMSL7, PvMSL8 and PvMSL9) (Figure 1B).In Class II, C-terminal TM domain proteins are not glycine-or alanine rich; however, they contain amino acids with larger hydrophobic side chains.Large hydrophobic amino acids are conserved at certain positions within the domain.In agreement with a previous analysis by Pivetti et al. (2003), the conserved motif includes most C-terminal TM domain and surrounding sequence in E. coli.Generally, members of MscS family have good conservation throughout the TM3 helix (Pivetti et al., 2003;Miller et al., 2003;Saddhe and Kumar, 2015).

Protein properties and subcellular localizations of PvMSL proteins
The parameters used to describe PvMSL proteins are listed in All putative PvMSL proteins were found in the membrane.Class I PvMSL genes (PvMSL1-PvMSL4) were localized either in the plasma membrane or chloroplast membrane, whereas Class II PvMSL genes (PvMSL5 -PvMSL9) were present only in the plasma membrane (Table 2).Consistently with studies in A. thaliana, AtMSL2 and AtMSL3 genes were localized in the plastid envelope, most likely in the inner membrane, and are observed in foci at the plastid poles (Haswell and Meyerowitz, 2006;Wilson et al., 2011).MSL genes of Class I were reported to be located either in the plastid envelop or mitochondria (Haswell, 2007;Saddhe and Kumer, 2015).Class II genes were found in the plasma membrane.Saddhe and Kumer (2015) showed that all Class II members of O. sativa (OsMSL4 -OsMSL7) are located in the plasma membrane.AtMSL9 and AtMSL10 genes in A. thaliana were found in the plasma membrane of root cells (Haswell et al., 2008).Immunofluorescence of algal Chlamydomonas reinhardtii similarly revealed a complex localization pattern for MSC1 genes located in the chloroplast and cytoplasm (Nakayama et al., 2007).The predicted topology of PvMSL genes were illustrated in Figure 2. In Class I, PvMSL1 and PvMSL3 proteins demonstrated the presence of five transmembrane (TM) regions, while PvMSL2 and PvMSL4 exhibited the presence of six and seven TM regions, respectively.In Class II, the number of TM regions ranged from six or seven.PvMSL6, PvMSL7 and PvMSL9 proteins exhibited the presence of six TM, while PvMSL5 and PvMSL8 showed the presence of seven TM regions.In E. coli, MscS channel topologies varied from 3 to 11 putative transmembrane segments (TMS) and adopted an Nterminal out and C-terminal in configuration (Miller et al., 2003;Pivetti et al., 2003).Indeed, the results identified in this study were similar to the ones in Arabidopsis and Rice Class I members which showed the presence of five TM domains while Class II members exhibited the presence of six TM domains (Haswell, 2007;Saddhe and Kumer, 2015).

Phylogenetic analyses and identification of conserved domains of PvMSL family
To clarify the phylogenetic relationships among MSL gene family, a phylogenetic tree was constructed based on the alignment of Arabidopsis, rice and common bean of MSL proteins sequences (Figure 3).The Maximum-Likelihood (ML) phylogenetic tree divided MSL genes into two classes (Figures 3 and 4), supported by bootstrap values and the occurrence of conserved protein motifs (Figure 4A and B).In Arabidopsis and rice, two classes, I and II, have been classified (Haswell and Meyerowitz, 2006;Saddhe and Kumar, 2015) and the corresponding groups in common bean are labeled in Figures 3 and 4A.The Class I and II common bean contains four and five PvMSL genes, respectively, according to the classification in Arabidopsis.The phylogenetic analysis revealed that common bean MSL genes showed similar phylogenetic relationships with Arabidopsis and rice.Among these, Class II constituted the largest clade, containing 15 members and accounting for 60% of total MSL genes, while Class I contained 10 members and accounted for 40% of total MSL genes.Additionally, in rice, a monocotyledon, the number of members within the two classes was the same, while in dicotyledon, Arabidopsis and common bean, the number of members was larger, indicating that these genes could have originated from a common ancestor by frequent duplication gene after the split between mono and dicotyledon.
To further reveal the diversification of MSL genes in common bean, putative motifs were predicted using the program MEME, and 10 distinct motifs were identified (Figure 4B).The schematic distribution of 10 motifs among the two gene classes is shown in Figure 4B; these motifs are represented in their relative location within the protein.The identified multilevel consensus sequence for the motifs is shown in Table 3.The 10 motifs identified by MEME were annotated by InterProScan.The superfamily database of structural and functional protein annotations showed that MS channel and like-Sm (LSM) domains were present in all common bean PvMSL protein sequences.The Pfam analysis showed that the MS channel domains were present in all PvMSL proteins.Protein information resource (PIR) database showed that common bean MSL, PvMSL5 to PvMSL9 have a unique MscS-like plant/fungus domain which is restricted to plant and fungus lineages.Motif 2 is a frequently occurring sequence and is present in most PvMSL genes, consisting of MS channel domain.As expected, most of the closely related members in the phylogenetic tree had common motif compositions, suggesting functional similarities among MSL proteins within the same class (Figure 4B).For example, motifs 2, 7 and 9 were specific to Class I, while motifs 3, 4, 5, 7, 8 and 10 were specific to Class II.Motifs3, 4 and 5 showed a casein kinase C phosphorylation site.All members of Class II(PvMSL5 to PvMSL9) were located in plasma membrane, which might be regulated by phosphorylation during protein kinase C.These similarities in motif patterns might be related to similar functions of MSL proteins within the same class.

Structural analyses, genome distribution, duplications and synteny of PvMSL genes
It is well known that gene structural diversity is a possible mechanism for the evolution of multigene  3. families.In order to gain further insight into the structural diversity of MSL genes, the exon/intron structure of each member of PvMSL family in common bean was analyzed.A detailed illustration of exon/intron structures is shown in Figure 5.According to their predicted structures, all PvMSL genes have introns in their structure and the number of exons varied from 4 to 12.These exon/intron structures are similar to rice (Saddhe and Kumer, 2015).
The most closely-related members in the same class generally showed the same exon/intron pattern in which the number, position and length of the intron almost completely conserved within classes (Figure 5).For instance, the gene structure appeared to be more variable in PvMSL genes of Class I, where the number of introns ranged from 5 to 12, while the members of Class II contained only four or five introns.Most genes containing introns were clustered into the same class suggesting evolutionary conservation in P. vulgaris.
The chromosomal locations of PvMSLs were determined based on the information from Phytozome Database of P. vulgaris.Genome chromosomal location analyses revealed that PvMSL were randomly distributed in 5 out of 11 chromosomes (Figure 6).Among these chromosomes, chromosome 9 contained the largest number of MSL genes, while the other chromosomes contain only one MSL gene.PvMSL2, PvMSL9, PvMSL5 and PvMSL1 genes were localized in chromosomes 2, 3, 7 and 10, respectively, as shown in Figure 6.Five genes, PvMSL3, PvMSL4, PvMSL6, PvMSL7 and PvMSL8, are located in chromosome 9. Substantial clustering of PvMSL genes was evident in chromosome 9, with high densities of the genes.
Tandem and segmental duplications have been suggested to be two of the main causes for gene family expansion in plants (Cannon et al., 2004).Two or more genes located in the same chromosome are confirmed as a tandem duplication event, while gene duplication between different chromosomes is designated as a segmental duplication event (Liu et al., 2011;Cai et al., 2013).Two pairs of MSL paralogues genes were identified in chromosomes 2, 3 and 9. Segmental duplications with high similarity (55%) were detected between PvMSL2 in the terminal region of chromosome 2 and PvMSL3 in the same region of chromosome 9.
PvMSL9 in chromosome 3 also showed a probable (50%) duplicated region shared with PvMSL8 in chromosome 9, both of which are located in the lower arm of chromosomes.According to Holub (2001), tandem duplications are 200 kb regions in a chromosome that contains two or more genes with high similarity.Therefore, two MSL tandem duplication clusters were identified.Four genes (PvMSL3, PvMSL4, PvMSL7, and PvMSL8) were arranged in two clusters in 7.7 and 33.7 kb segments in chromosome 9, respectively (Figure 6).This strongly suggested that they might be generated by tandem duplications.The duplication prediction analysis indicated that the evolution of PvMSL gene family in common bean genome resulted from a total of 4 gene duplications, 2 segmental and 2 tandem duplication events (Figure 6).
In our study, one comparative syntenic map of P. vulgaris associated with A. thaliana (Figure 7) was built.A total of three pairs of syntenic relations were identified, three AtMSL genes (AtMSL1, AtMSL7 and AtMSL10) and three PvMSL genes (PvMSL1, PvMSL5 and PvMSL6) were found to be associated with synteny events.These synteny events suggest that many MSL genes arose before the divergence of Arabidopsis and common bean lineages.Thus, this result may indicate that PvMSL genes in P. vulgaris share similar structure and function with AtMSL genes in Arabidopsis.

Expression profiles of PvMSL genes
It has been noted that MSL gene family exhibits differences among different organisms and different tissues to exert different physiological functions.Thus, gene expression patterns can provide important clues for gene function.To further analyze the tissue specificity of MSL gene family members, the expression profiles of the nine PvMSL genes were analyzed as shown in Figure 8.Most of MSL genes showed distinct tissue-specific expression patterns across the nine examined tissues.Some of them were constitutively expressed in almost all tissues and organs, and the expression levels were high, such as those of PvMSL1, PvMSL2, PvMSL3 and PvMSL6.The expression levels of PvMSL1, PvMSL2 and PvMSL3 were relatively higher in leaves and roots, which indicates that they could play a role in the development of plant root and leaves.These expression patterns were similar to AtMSL9 and AtMSL10 in Arabidopsis, which are expressed at high levels in root and responded to mechanical and gravity stimuli (Haswell, 2007;Kimbrough et al., 2004;Haswell et al., 2008).Likewise, AtMSL2 and AtMSL3 were found in leaves and control plastid size, shape, and perhaps division during normal plant development by altering ion flux in response to changes in membrane tension (Haswell and Meyerowitz, 2006).In eukaryotic, MSL genes presented varied distribution and function, in addition to serving as a safety valve (Saddhe and Kumar, 2015).PvMSL6 and PvMSL9 were relatively higher expression in nodules and young trifoliates, respectively.PvMSL4, PvMSL7 and PvMSL8showed very low expression in almost all tissues and organs of common bean.Detailed analysis of the expression patterns of PvMSL showed that some of the genes clustered in the same class of the phylogenetic tree (Figure 4) had similar expression patterns, also indicating the existence of redundancy among MSL genes in this class.For example, most PvMSLs in Class I were mainly expressed in leaves and roots while all genes in Class II were little expressed in different tissues and organs of common bean.In addition, paralogous PvMSL genes, such as PvMSL2-PvMSL3 and PvMSL8-PvMSL9, revealed distinct expression patterns.These expression profiles suggest a divergence in the biological functions of PvMSL genes during plant development.

Conclusion
Based on in silico approach, this is the first study that provides some information about MSL family in P. vulgaris and has a crucial role in the plant growth and development.
Understanding the genetic bases of MS ion channels provide resources to select candidate genes for future functional analyses of PvMSL family in common bean, increasing the possibility to genetically engineer new traits of importance for agriculture and food production.

Figure 1 .
Figure 1.Alignment of MSL family conserved domains.Amino acid sequence of MSL family members from A. thaliana (AtMSL1 to AtMSL10), O. sativa (OsMSL1 to OsMSL6) and P. vulgaris (PvMSL1 to PvMSL9).Consensus sequences derived from this analysis is presented at the bottom of each alignment in dark type; the consensus sequence derived by Pivetti et al. (2003) is below and of each alignment in gray type.(A) Class I proteins.The box indicate the experimentally derived MscS TM3 domain.(B) Class II proteins.The box indicate the location of a consensus TM3 domain.
, the eukaryotic members of MscS family can be organized into two main classes based on sequence similarity.Proteins in Class I have transmembrane (TM) domains at the C-terminal region that resemble TM3 MscS which are rich in glycine and alanine residues, though the pattern is not conserved (Figure 1A).Conserved motif PF(X12-16)GXV(X20-21)PN(X9)N was identified at C-terminal for the TM domain in Class I MSL in common bean (PvMSL1, PvMSL2, PvMSL3 and PvMSL4).Class I proteins align relatively closely to E. coli MsSC and their C-terminal TM domains

Figure 2 .
Figure 2. A cartoon of the transmembrane (TM) helix topology summarizing the linear coordinates for the helices and indicating the protein's extra-and intercellular regions.Transmembrane helix region represented by the yellow boxes labeled S1, S2, S3, S4, S5, S6 and S7.Membrane regions were represented by the black boxes.N and C terminal regions were labeled and represented as a single line.

Figure 3 .
Figure 3. Phylogenetic tree of A. thaliana, O. sativa, and P. vulgaris MSL genes.The tree was based on 25 MSL sequences (including both N-terminal and C-terminal domains) from three species using the Maximum-Likelihood method.

Figure 4 .
Figure 4. Relationships and schematic representation of motif compositions of 9 members of bean MSL family.Unrooted ML phylogenetic tree was based on nine PvMSL protein sequences using MEGA6.06.The motifs identified by MEME software are represented by colored boxes and their consensus sequences are shown in Table3.

Figure 5 .
Figure 5. Gene structure schematic diagram for PvMSL genes.Exons were demonstrated by filled yellow boxes and introns were demonstrated by black lines.Untranslated region (UTR) was displayed by blue boxes at both ends.

Figure 6 .
Figure 6.Chromosomal location of PvMSL The chromosome number is indicated at the top of each linkage group.The chromosomal positions of putative PvMSL were mapped according to genome coordinates.

Figure 7 .
Figure 7. Synteny map showing the orthologous gene positions of MSL genes between Phaseolus vulgaris and Arabidopsis.P. vulgaris (Pv07, Pv09 and Pv10) and Arabidopsis chromosomes (At02, At04 and At05) maps were based on the orthologous and demonstrate highly conserved synteny.Each block represents individual chromosome and red lines connecting two chromosomal regions denote syntenic regions of genomes.

Figure 8 .
Figure 8. Differential expression patterns of common bean PvMSL genes.Heat map showing differential expression profile of common bean PvMSL genes inspecifictissues: leaves, roots, nodule, young trifoliates, flower buds, stem, flowers, green mature pods and young pods.

Table 1 .
Comparison of number of members of each MSL class of genes between species Phaseolus vulgaris, Arabidopsis thaliana, Glycine max, Solanum lycopersicum, Oryza sativa and Zea mays, respectively.
a Means motif ID.